Technical Abstract:
Previous study has shown that IgE binding of peanut major allergens can be reduced by cross-linking of their tyrosine residues induced by peroxidase. Because tyrosine is vulnerable to metal-catalyzed oxidation, we determined if metals can catalyze cross-linking, and thus, reduce IgE binding of peanut allergens. Peanut extracts, pH 8, were incubated for various times at 37 oC with copper sulfate (CuSO4) or ferric chloride (FeCl3) in the presence of hydrogen peroxide. The metal-treated extracts were then analyzed by SDS-PAGE for allergen cross-links. Monoclonal antibodies against dityrosine were also used to confirm the presence of cross-links. Changes of IgE binding were measured in Western blots and competitive ELISA, using a pooled serum from peanut-allergic individuals. Results showed that of the two metals tested, only Cu(II) had an effect on Ara h 1 and Ara h 2 allergens. In the presence of hydrogen peroxide, and at >5 hr, Cu(II) induced a reduction in the allergen levels, plus the formation of allergen cross-links detected in SDS-PAGE. Cross-links were also detected by monoclonal antibodies against dityrosine in Western blots. Only allergens from roasted peanuts were affected. Cross-links bound to IgE to some extent in Western blots. Despite this, IgE binding of Cu-treated extracts was significantly lower than that of the untreated in competitive ELISAs. We concluded that treatment of roasted peanut extracts with Cu(II), in the presence of hydrogen peroxide, resulted in a reduced level of Ara h 1/ Ara h 2, and the formation of allergen cross-links. No effect was seen with Fe(III). IgE binding of Cu-treated extracts was significantly reduced as compared to the untreated. This suggests that metals, such as copper, can be used to reduce the allergenicity of peanut allergens.